Glucan composition and process for preparation thereof

ABSTRACT

Three dimensional glucan matrix compositions are prepared by separating growing yeast from its growth medium, subjecting the yeast with cell walls intact to an alkali material, thereby extracting whole glucan particles having an intact cell wall structure. The whole glucans can then, optionally, be treated with acetic acid to alter the β(1-6) linkages, or with glucanase to alter the β(1-3) linkages. The glucans have viscosity characteristics dependent upon the strain of yeast utilized and are useful as stabilizers or thickeners.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Ser. No. 675,927,filed Nov. 28, 1984 now U.S. Pat. No. 4,810,646, entitled "GlucanCompositions And Process For Preparation Thereof" by S. Jamas, C. K. Rhaand A. J. Sinskey.

FIELD OF INVENTION

The present invention relates to biopolymer engineering.

BACKGROUND OF THE INVENTION

The food industry uses many naturally derived polysaccharides asstabilizers and thickeners. Other industries use polysaccharides aswater treatment chemicals, viscosifiers, thickeners and as surfaceactive materials. Products such as carrageenan, alginate and starch,exhibit unique structural and rheological properties such as yieldstress and an ability to increase the viscosity in an aqueousenvironment. The specific structure-function relationships of thesebiopolymers depend on the individual components such as monomers orrepeating units and their chemical linkages.

Polysaccharides which form the bulk of biopolymers in the microbialworld have already been noted for their structural importance and areresponsible for maintaining the integrity of bacteria and fungi. Withthe advent of genetic engineering, biosynthesis and manufacture of thesebiopolymers can be directed to produce molecules with altered physicalproperties.

Yeast has historically earned its role as an important food grade andindustrial organism. The cell wall of Saccharomyces cerevisiae is mainlycomposed of β-linked glucan. This polymer is responsible for the shapeand mechanical strength of the cell walls. The glucan is mainly composedof a backbone chain of β(1-3) linked glucose units with a low degree ofinter and intra-molecular branching through β(1-6) linkages. A minorcomponent that consists mainly of a highly branched β(1-6) linked glucanis closely associated with the main component and both comprise thealkali insoluble glucan fraction.

The following articles deal with the structure of glucans: "TheStructure of a β-(1-6)-D-Glucan from Yeast Cell Walls", by Manners etal., Biochem. J., (1973) 135:31-36; "Evidence for Covalent Linkagesbetween Chitin and β-Glucan in Fungal Wall", by Sietsma et al., Journalof General Microbiology (1979), 114:99-108; "Demonstration of aFibrillar Component in the Cell Wall of the Yeast SaccharomycesCervisiase and its Chemical Nature", by Kopecka et al., The Journal ofCell Biology, 62 (1974), 66-76; "On the Nature and Formation of theFibrillar Nets Produced by Photoplasts Saccharomyces Cerevisiae inLiquid Media: An Electronmicroscopic, X-Ray Diffraction and ChemicalStudy", by Kreger et al., Journal of General Microbiology, (1975),92:202-220; "Short Communication Solubility of(1-3)-β-D-(1-6)-β-D-glucan in Fungal Walls: Importance of PresumedLinkage between Glucan and Chitin", by Sietsma and Wessels, Journal ofGeneral Microbiology, (1981), 125:209-212; "The Molecular ConstitutionOf An Insoluble Polysaccharide From Yeast, Saccharomyces cerevisiae", byHassid et al. Journal of the American Chemical Society, 63:295-298(1941); "Comparative Tumor-Inhibitory and Anti-Bacterial Activity ofSoluble and Particulate Glucan", by DiLuzio et al., InternationalJournal of Cancer, 24:773-779 (1979).

SUMMARY OF THE INVENTION

By processing yeast cells and the glucans derived therefrom according tothe techniques of the present invention, a glucan product which retainsthe three dimensional morphology of the intact yeast cell wall andhaving high water holding capacity is formed, which in turn may befurther processed to give glucans having improved or novel functionalproperties.

In one embodiment of the present invention, there is provided a glucanderived from yeast which retains the intact cell wall structure of theyeast cell in vivo. Glucan particles having these properties is referredto as "whole glucan particles". A process for producing said glucan isalso described. The process produces whole glucan which has a high levelof purity, and consistency of the size and shape of the particle.

Whole glucan particles may be obtained from any glucan-containingsource, including yeast or other fungi. The yeast is preferably a strainof Saccharomyces cerevisiae, but any strain of yeast can be used. Thesepure whole glucan particles are typically spherical, and exhibit a highwater holding capacity, as exhibited by their viscosity in aqueoussolutions. For example, an aqueous suspension of whole glucan particlesderived from strain Saccharomyces cerevisiae A364A, having a particlesize of approximately 2 to approximately 4 microns containing about 5.5grams of glucan per deciliter has a viscosity of about 1000 centipoise.A Saccharomyces cerevisiae 374 derived glucan, having a particle size offrom about 2.5 to about 6.3 microns, has a viscosity of about 2630centipoise in an aqueous suspension containing about 3.5 grams of glucanper deciliter.

Further, in another embodiment of the present invention, there isprovided aqueous hydroxide insoluble whole glucan particles derived froma mutant strain of yeast which has altered β(1-6) linkages for enhancingthe structural, biological and physical properties of the material. Forexample, the water holding capacity of whole glucan derived from thesemutants by the present process is significantly enhanced. A method forproducing the mutant yeast strains is provided.

Also provided is a glucan having altered β(1-6) or altered β(1-3)linkages for controlling the physical properties of the glucanparticles, and methods for chemically altering the β(1-6) and β(1-3)linkages. The amount of β(1-6) linked glucan can be decreased bytreating the whole glucan with an acid (e.g., acetic acid). Decreasingthe β(1-6) linkages yields whole glucan particles with a higher waterholding capacity, as evidenced by the greater aqueous viscosity of asuspension of the acid-modified whole glucan particles. Treating thewhole glucan particles with a hydrolytic glucanase enzyme (e.g.laminarinase) decreases the amount of β(1-3) linked glucan which yieldswhole glucan particles having a lower water holding capacity, asevidenced by the reduced aqueous viscosity of a suspension of theglucanase-modified whole glucan particles.

The term "altered" as used herein and applied to the structure of theglucan (i.e., the β(1-6) or β(1-3) linkages) shall be construed to meanthat the glucan structure has been modified or changed in some way,endowing the altered glucan with properties which are measurablydifferent from those of naturally occurring unmodified glucans.

The modified glucans set forth above can be produced by processesdescribed hereinafter.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph showing a plot of the viscosity profile of whole yeastglucan for Saccharomyces cerevisiae 374, 377 and A364A.

FIG. 2 is a graph showing a plot of the network-compression modulus vs.volume fraction for suspensions of whole glucan derived fromSaccharomyces cerevisiae A364A and R4.

FIG. 3 is a graph showing a plot of the viscosity profile of wholeglucan derived from Saccharomyces cerevisiae A364A after 4 hours oflaminarinase digestion.

DETAILED DESCRIPTION OF THE INVENTION

The process described below for producing the glucan particles can beseparated into two steps. The first step involves the extraction andpurification of the alkali-insoluble whole glucan particles from theyeast or fungal cell walls. This process yields a product whichmaintains the morphological and structural properties of the glucan asfound in vivo and will be referred to as a whole glucan, or whole glucanparticles.

The second step, which is optional, involves the modification of thestructure of the whole glucan particles by chemical or enzymatictreatment. In this step, the structure-function properties of the wholeglucan particles obtained from the first step can be altered or modifiedin a controllable manner. For example, the the ratio of β(1-6)/β(1-3)linkages can be chemically or enzymatically adjusted, thereby causing acommensurate change in the structural rigidity, hydrodynamic propertiesand biological properties of the whole glucan particles. The change inthe hydrodynamic properties of the modified whole glucan particles canbe evidenced by the change in the viscosity profile of an aqueoussolution of the glucan particles. In this step, the amount of β(1-6)linked glucan can be decreased by treating the whole glucan with acid(e.g., acetic acid) or with a β(1-6) specific glucanase enzyme. Theamount of β(1-3) linked glucan can be decreased by treating the wholeglucan particles with a β(1-3) specific glucanase enzyme (e.g.,laminarinase) or, alternatively, hydrolyzing with acids. Using thesemethods, the structure/function properties of the whole glucan particlescan be controlled.

The structure-function properties of the whole glucan preparationdepends directly on the source from which it is obtained. The source ofwhole glucan can be yeast or other fungi, or any other source containingglucan having the properties described herein. Yeast cells are apreferred source of glucans. The yeast strains employed in the presentprocess can be any strain of yeast, including, for example,Saccharomyces cerevisiae, Saccharomyces delbrueckii, Saccharomycesrosei, Saccharomyces microellipsodes, Saccharomyces carlsbergensis,Saccharomyces bisporus, Saccharomyces fermentati, Saccharomyces rouxii,Schizosaccharomyces pombe, Kluyveromyces polysporus, Candida albicans,Candida cloacae, Candida tropicalis, Candida utilis, Hansenula wingei,Hansenula arni, Hansenula henricii, Hansenula americana, Hansenulacanadiensis, Hansenula capsulata, Hansenula polymorpha, Pichia kluyveri,Pichia pastoris, Pichia polymorpha, Pichia rhodanensis, Pichia ohmeri,Torulopsis bovina, and Torulopsis glabrata. Mutants prepared from theseor other yeast stains, can also be used, depending upon the biologicaland hydrodynamic properties desired. The yeast strain may be mutated, aswill be described below, to affect the glucan structure and, hence, itsproperties. For example, mutations can be induced by which the relativeamount of β(1-6) or β(1-3) linkages may be increased, or reduced,thereby causing a commensurate change in the structural rigidity, andthe biological and hydrodynamic properties of the whole glucanparticles. Saccharomyces cerevisiae and mutants therefrom are preferredstrains. In particular, Saccharomyces cerevisiae A364A and its twotemperature sensitive, cell division cycle mutants, Saccharomycescerevisiae 374 and 377. The strain Saccharomyces cerevisiae R4 is amutant of A364A which has been isolated on the basis of an increasedβ(1-6) glucan fraction. The R4 mutant can also be employed to obtainglucan matrices with altered structure-function properties.

The following procedure was employed to prepare and isolate mutant R4,which has an increased number of β(1-6) linkages, and can be used toprepare and isolate other yeast strains having altered β(1-6) or β(1-3)linkages:

Mutant strain R4 was derived from Saccharomyces cerevisiae A364A. Theparent strain, A364A, was grown in YPD to mid-log phase. The cells werewashed and divided into aliquots in sterile glass petri plates. Thecells were exposed to a suitable mutagen, which in this case was UVexposure of 25 sec (30% survival). The cells were then suspended in YPDand grown under subdued light to a concentration of approximately 5×10⁶CFU/ml. The cells were harvested, and protoplasts were prepared bydigesting the cells with 0.25 mg/ml of a glucanase enzyme, in this caselaminarinase, for 30 minutes. Other hydrolytic enzymes which willdegrade the cell wall can also be used. The suspension was then dilutedwith water to lyse osmotically sensitive protoplasts. The survivingcells then were grown in YPD to a density of approximately 5×10⁶ CFU/ml(approximately 10 hours). Treatment with laminarinase, followed bygrowth was repeated two more times using 1.0 mg/ml enzyme for 15minutes. The candidates which showed resistance to laminarinasedigestion were then streaked on YPDA plates. Single colony isolates weretested for resistance to the enzyme compared to the resistance of theparent strain, A364A. The mutant R4 is available from the AgriculturalResearch Service under No. NRRL Y-15903.

The above procedure can be used to prepare and isolate other mutantyeast strains by using other parent strains as starting material. Othermutagens can be employed to induce the mutations, for example, chemicalmutagens, irradiation, or other DNA and recombinant manipulations. Otherselection or screening techniques may be similarly employed.

The yeast cells may be produced by methods known in the art. Typicalgrowth media comprise, for example, glucose, peptone and a yeastextract. The yeast cells may be harvested and separated from the growthmedium by methods typically applied to separate the biomass from theliquid medium. Such methods typically employ a solid-liquid separationprocess such as filtration or centrifugation. In the present process,the cells are preferably harvested in the mid-to late logarithmic phaseof growth, to minimize the amount of glycogen and chitin in the yeastcells. Glycogen, chitin and protein are undesirable contaminants whichaffect the biological and hydrodynamic properties of the whole glucanparticles.

The herein referred to first step according to the process of thepresent invention, involves treating the yeast with an aqueous alkalinesolution at a suitable concentration to solubilize a portion of theyeast and form an alkali-hydroxide insoluble whole glucan particleshaving primarily β(1-6) and β(1-3) linkages. The alkali generallyemployed is an alkali-metal hydroxide, such as sodium or potassiumhydroxide. Preferably, the starting material consists essentially ofyeast separated from the growth medium. It is more difficult to controlconsumption of the aqueous hydroxide reactants and the concentration ofreactants in the preferred ranges when starting with yeast compositionsthat are less concentrated. The yeast should have intact, unrupturedcell walls since the preferred properties of the instant whole glucanparticles depend upon an intact cell wall.

The treating step is performed by extracting the yeast in the aqueoushydroxide solution. The intracellular components and mannoproteinportion of the cell are solubilized in the aqueous hydroxide solution,leaving insoluble cell wall material which is substantially devoid ofprotein and having a substantially unaltered three dimensional matrix ofβ(1-6) and β(1-3) linked glucan. The preferred conditions of performingthis step result in the mannan component of the cell wall beingdissolved in the aqueous hydroxide solution. The intracellularconstituents are hydrolyzed and released into the soluble phase.Preferably, the conditions of digestion are such that at least in amajor portion of the cells, the three dimensional matrix structure ofthe cell walls is not destroyed. More preferably, substantially all thecell wall glucan remains unaltered and intact.

The aqueous hydroxide digestion step is preferably carried out in ahydroxide solution having initial normality of from about 0.1 to about10.0. Typical hydroxide solutions include hydroxides of the alkali metalgroup and alkaline earth metals of the Periodic Table. The preferredaqueous hydroxide solutions are of sodium and potassium, due to theiravailability. The digestion is preferably carried out at a temperatureof from about 20° C. to about 121° C. with lower temperatures requiringlonger digestion times. When sodium hydroxide is used as the aqueoushydroxide, the temperature is preferably from about 80° C. to about 100°C. and the solution has an initial normality of from about 0.75 to about1.5. The hydroxide added is in excess of the amount required, thus, nosubsequent additions are necessary.

From about 10 to about 500 grams of dry yeast per liter of hydroxidesolution is used. Preferably the aqueous hydroxide digestion step iscarried out by a series of contacting steps so that the amount ofresidual contaminants such as proteins are less than if only onecontacting step is utilized. In other words, it is desirable to removesubstantially all of the protein material from the cell. Preferably suchremoval is carried out to such an extent that less than one percent ofthe protein remains with the insoluble cell wall glucan particles. Anadditional extraction step is preferably carried out in a mild acidsolution having a pH of from about 2.0 to about 6.0. Typical mild acidsolutions include hydrochloric acid, sodium chloride adjusted to therequired pH with hydrochloric acid and acetate buffers. This extractionstep is preferably carried out at a temperature of from about 20° C. toabout 100° C. The digested glucan particles can be, if necessary,subjected to further washings and extraction to reduce the protein andcontaminant level to the preferred amounts hereinbefore indicated.

By conducting this process without a step of disrupting the cell walls,the extraction can be conducted at more severe conditions of pH andtemperature than was possible with the prior art procedure whichincluded a step of disrupting the cell walls. That is, the process ofthis invention avoids product degradation while employing these severeextraction conditions which permits elimination of time-consumingmultiple extraction steps.

After the above aqueous hydroxide treatment step, the final whole glucanproduct comprises about 10 to about 15 percent of the initial weight ofthe yeast cell, preferably the product is from about 12 to about 14percent by weight.

The aqueous hydroxide insoluble whole glucan particles produced is asset forth in the summary of the invention. The whole glucan particlescan be further processed and/or further purified, as desired. Forexample, the glucan can be dried to a fine powder (e.g., by drying in anoven); or can be treated with organic solvents (e.g., alcohols, ether,acetone, methyl ethyl ketone, chloroform) to remove any traces ororganic-soluble material, or retreated with hydroxide solution, toremove additional proteins or other impurities which may be present.

The whole glucan particles obtained from the present process arecomprised of highly pure glucan, which consists essentially of β(1-6)and β(1-3) linked glucan. The whole glucan particles contain very littlecontamination from protein and glycogen. Preferably, the whole glucanparticles are spherical in shape with a diameter of about 2 to about 4microns and contain greater than 85% by weight hexose sugars,approximately 1% by weight protein and no detectable amount of mannan,as determined by Fourier Transform Infrared Spectroscopy. Glucansobtained by prior processes contain substantially higher quantities ofchitin and glycogen than the present glucans.

The second step as set forth above, involves the modification of thewhole glucan particles, as produced above, by chemical treatment tochange the properties of the glucan. It is contemplated that wholeglucan particles, derived from any yeast strain may be used, in additionto those particular strains described herein. As mentioned above, a verybroad spectrum of yeast strains may be used. The processing conditionsdescribed above are also applicable to glucan extraction from fungi ingeneral. The properties of these glucans also will depend on the sourcesfrom which they are derived.

According to a first chemical treatment, the whole glucan particles canbe treated with an acid to decrease the amount of β(1-6) linkages andthus, change the hydrodynamic properties of said glucans as evidenced byan increase in the viscosity of aqueous solutions of these modifiedglucans.

In accordance with the principles of the present invention, there isprovided a process for preparing an altered whole glucan particles bytreating the glucan particles with an acid, for a suitable period oftime to alter the β(1-6) linkages. Acetic acid is preferred, due to itsmild acidity, ease of handling, low toxicity, low cost and availability,but other acids may be used. Generally these acids should be mild enoughto limit hydrolysis of the β(1-3) linkages. The treatment is carried outunder conditions to substantially only affect the β(1-6) linked glucans.Preferably, the acid treatment is carried out with a liquid consistingessentially of acetic acid, or any dilutions thereof (typical diluentscan be organic solvents or inorganic acid solutions). The treatment ispreferably carried out at a temperature of from about 20° C. to about100° C. Preferably, the treatment is carried out to such an extent toremove from about 3 to about 20 percent by weight of acid solublematerial based on total weight of the whole glucan particles beforetreatment. More preferably, the extent of removal is from about 3 toabout 4 percent by weight. The preferred compositions formed demonstratealtered hydrodynamic properties and an enhancement in viscosity aftertreatment.

According to a second chemical treatment, the whole glucan particles aretreated with an enzyme or an acid, to change the amount of β(1-3)linkages. For whole glucan particles derived from some yeast strains,enzyme treatment causes a decrease in the viscosity, and for others, itcauses an increase in viscosity, but in general, alters the chemical andhydrodynamic properties of the resulting glucans. The treatment is witha β(1-3) glucanase enzyme, such as laminarinase, for altering the β(1-3)linkages to alter the hydrodynamic properties of the whole glucanparticles in aqueous suspensions.

The enzyme treatment can be carried out in an aqueous solution having aconcentration of glucan of from about 0.1 to about 10.0 grams per liter.Any hydrolytic glucanase enzyme can be used, such as laminarinase, whichis effective and readily available. The time of incubation may varydepending on the concentration of whole glucan particles and glucanaseenzyme. The β(1-3) linkages are resistant to hydrolysis by mild acidssuch as acetic acid. Treatment with strong or concentrated acids, suchas hydrochloric acid (HCl), sulfuric acid (H₂ SO₄) or formic acid,hydrolyzes the β(1-3) linkages thereby reducing the amount of β(1-3)linkages. The acid treatment can be carried out in an aqueous solutionhaving a concentration of glucan from about 0.1 to about 10.0 grams perliter. The time of acid treatment may vary depending upon theconcentration of whole glucan particles and acid. Acid hydrolysis can becarried out at a temperature of from about 20° C. to about 100° C. Thepreferred compositions formed demonstrate altered hydrodynamicproperties.

By controlling the incubation time, it is possible to control thechemical and hydrodynamic properties of the resulting product. Forexample, the product viscosity can be precisely controlled forparticular usage, as, for example, with a variety of food products.

A hydrodynamic parameter (K₁) of the final treated product havingaltered linkages is dependent on the treatment time according to thefinal formula:

    K.sub.1 =-0.0021 (time)+0.26

where time is in minutes

where time is less than one hour.

The parameter K₁ is directly related (proportional) to the relativeviscosity. In the case of aqueous suspensions the relative viscosity isequal to the actual viscosity when the latter is measured in centipoise.

A process for preparing an aqueous slurry of a glucan having apredetermined desired viscosity is provided. The slurry comprises glucanat a concentration which is a function of the predetermined desiredviscosity according to the following approximate formula: ##EQU1##where, K₁ =(shape factor)×(hydrodynamic volume)

K₂ =(hydrodynamic volume)/(maximum packing fraction)

The shape factor is an empirically determined value which describes theshape of the glucan matrix in its aqueous environment. The shape factoris a function of the length:width ratio of a particle and can bedetermined microscopically. The hydrodynamic volume is a measure of thevolume a particle occupies when in suspension. This is an importantparameter for glucan suspensions at it indicates the high water holdingcapacity of glucan matrices. The maximum packing fraction can bedescribed as the highest attainable volume fraction of glucans which canbe packed into a unit volume of suspension.

The invention is further illustrated by the following examples.

EXAMPLE 1

In this example, whole glucan is prepared from Saccharomyces cerevisiaeA364A, 374, 377 and R4. A 14 liter Chemap fermenter was used to producethe biomass in 10 liters of growth medium which comprised 2% glucose, 2%peptone and 1% yeast extract. The fermenter was inoculated with 250 mlof a stationary phase culture and operated at a temperature of about28°-30° C., 400 rpm impeller speed, 1 vvm aeration rate and a pH of5.5±0.1. The growth of the cells was followed by removing 10 millilitersamples and measuring the turbidity in a Klett-Summerson colorimeter. Inthe fermentation of strains 374 and 377 a temperature shift to 37° C.was performed at the early exponential growth phase. This was equivalentto a Klett reading of approximately 30 units.

The fermentation was stopped at the late exponential growth phase, whichcorresponds to approximately 120 Klett units.

The cells were harvested by batch centrifugation at 8000 rpm for 20minutes in a Sorval RC2-B centrifuge. The cells were then washed twicein distilled water in order to prepare them for the extraction of thewhole glucan. The first step involved resuspending the cell mass in 1liter 4% w/v NaOH and heating to 100° C. The cell suspension was stirredvigorously for 1 hour at this temperature. The insoluble materialcontaining the cell walls was recovered by centrifuging at 2000 rpm for15 minutes. This material was then suspended in 2 liters, 3% w/v NaOHand heated to 75° C. The suspension was stirred vigorously for 3 hoursat this temperature. The suspension was then allowed to cool to roomtemperature and the extraction was continued for a further 16 hours. Theinsoluble residue was recovered by centrifugation at 2000 rpm for 15minutes. This material was finally extracted in 2 liters, 3% w/v NaOHbrought to pH 4.5 with HCl, at 75° C. for 1 hour. The insoluble residuewas recovered by centrifugation and washed three times with 200milliliters water, once with 200 milliliters dehydrated ethanol andtwice with 200 milliliters dehydrated ethyl ether. The resulting slurrywas placed on petri plates and air dried at 37° C. for 12 hours to afine white powder. An example of the yields obtained from thisextraction and purification process is shown in Table 1:

                  TABLE 1                                                         ______________________________________                                        Yields of Whole Glucan Particles from                                         Saccharomyces cerevisiae A364A                                                Batch #           1         2      3                                          ______________________________________                                        Dry cell weight, DCW, (g)                                                                       14.03     15.25  9.78                                       Whole Glucan Particles (g)                                                                      1.86      1.64   1.53                                       % DCW extracted   13        11     15                                         Protein content.sup.1                                                                           0.73      0.81   0.88                                       (% w/w)                                                                       ______________________________________                                         .sup.1 As measured by Ninhydrin assay using lysine as a control.         

Although a small quantity of protein was detected in the whole glucanparticles, it was proven that the overwhelming component in thispreparation was β-glucan. The purity of this preparation was tested byobtaining infra-red spectra of the whole glucan samples. Samples wereprepared in solid KBr discs and analyzed in a Perkin-Elmer infra-redSpectrophotometer. The spectra obtained were compared with the spectrumof a standard β-glucan, Laminarin, purchased from Sigma ChemicalCompany. The whole glucan samples from all three strains gavecharacteristic spectra of glucan. All the peaks characteristic of theβ-glucan structural backbone at 7.95, 8.35, 8.7 and 11.3 m were obtainedfor all the whole glucan samples.

The whole glucan particles were then rehydrated in distilled water inorder to determine the viscosity profile of the suspension. Viscositywas measured using a Cannon-Fenske capillary viscometer (size 75). Theviscosity profiles of whole glucan particle suspensions for the threestrains are shown in FIG. 1.

The units of concentration in g/dl (g/100 milliliters) are equivalent to% w/v. By applying the linear model which was discussed in the detaileddescription of this disclosure, the relevant information concerning thehydrodynamic properties of the whole glucan particles was obtained. Theaccuracy of this model is reflected in the values of the regressioncoefficient, r, shown in Table 2:

                  TABLE 2                                                         ______________________________________                                        Hydrodynamic Properties of Whole Glucan Particles                                     Regression                                                                              Shape     Hydrodynamic                                      Glucan  Coefficient                                                                             Factor    Volume                                            Sample  r         v         v (dl/g)  φ.sub.m                             ______________________________________                                        A364A   0.9986    2.5       0.092     0.63                                      374   0.9987    4.1       0.088     0.36                                      377   0.9974    4.1       0.091     0.45                                    R4      0.9995    2.5       0.087      .66                                    ______________________________________                                         1. φ.sub.m = Maximum packing fraction                                

The whole glucan particles produced from mutant R4 has a similarviscosity profile to that of whole glucan from A364A, however, byincreasing the degree of β(1-6) crosslinking in vivo a glucan matrix hasbeen developed with a significantly higher mechanical strength as shownin Table 5. The strength (rigidity) of the glucan matrices was measuredusing centrifugal compression of the glucan matrices.

Table 3 illustrates the structural rigidity (elastic modulus) of wholeglucan matrices.

                  TABLE 3                                                         ______________________________________                                        The Elastic Modulus of Whole Glucan Matrices                                  Measured in the Range 0-30 g                                                         Network Modulus (Nm.sup.-2)                                            Source   φ = 0.048                                                                           0.050      0.052                                                                              0.054                                      ______________________________________                                        A364A     170       225        310  480                                       Whole                                                                         Glucan                                                                        R4       1300      2400       6000 42000                                      Whole                                                                         Glucan                                                                        ______________________________________                                    

Comparison With Glucan Prepared By Prior Art Method

Whole glucan produced by the above method was compared to glucanprepared by a prior art method.

Glucan material was prepared according to the procedure described inManners, et al., in Biochem. J., 135:31-36 (1973). This material wasthen used in a number of assays performed in parallel with glucansprepared by Applicants procedure from strains A364A and R4. Thefollowing data was obtained:

1. Protein content

2. Presence and level of glycogen

3. Relative levels of chitin

The functional component of the yeast cell wall in terms of solutionproperties and water-holding capacity is solely the beta-glucan. It istherefore important to minimize the levels of the above contaminants fortwo reasons: first, that the presence of hydrophobic contaminants suchas proteins and glycogen will hinder the functional properties of theglucan matrix; secondly, the purity of the preparation is an essentialfactor when considerating the application of glucans in both thepharmaceutical and food industries.

Protein content was determined chemically using the Bio-Rad assay(Bio-Rad). In this procedure, glucan samples were suspended in water toa concentration of 5 mg/ml, and lysozyme solutions in the range of 0.2to 1.0 mg/ml were prepared as standards. Duplicate 1.0 ml aliquots ofthe samples and standards were placed in clean dry test tubes and 5.0 mlof the undiluted dye reagent was added. The solutions were vortexed, andafter 5 minutes, the optical density (O.D.) was measured at 595 nm. Awater blank was used as a negative control.

Total hexose was measured in duplicate 100-fold dilutions of glucansuspensions. A standard curve was prepared using glucose solutions inthe range of 10-100 ug/ml. Duplicate 1.0 ml aliquots of the samples wereplaced in clean dry test tubes, and 1.0 ml of 5% (v/v) phenol was added.Then, 5 ml of concentrated sulfuric acid (H₂ SO₄) was added to eachtube, the mixture was vortexed, and incubated at 37° C. for 15-20minutes. The optical density was measured at 488 nm with a water sampleas the blank. The results are shown in Table 4.

                  TABLE 4                                                         ______________________________________                                        Protein Contamination of Glucan Particles                                                 Protein    Hexose                                                 Preparation mg/ml      mg/ml   Protein/Hexose                                 ______________________________________                                        A364A       0.071      8.74    0.0081                                         R4          0.082      8.81    0.0093                                         Manner's Prep                                                                             1.808      8.88    0.2036                                         ______________________________________                                    

Manner's preparation contained protein levels approximately 25 timeshigher than those of glucans prepared by the present procedure.

Fourier-Transform Infrared (FT-IR) spectroscopy was used to detect thepresence of chitin and glycogen, and peak integration was used todetermine their relative levels. The characteristic glycogen peaks whichappear at a wave number of 850, 930 and 760 cm⁻¹ were not detected inthe preparations made according to Applicants' procedure, but wereclearly present in Manners' preparation. In addition, the characteristicchitin absorbance at 1550 cm⁻¹ was significantly stronger for theManner's preparation. These results were quantitated and are summarizedin Table 5.

                  TABLE 5                                                         ______________________________________                                        Contaminating Polysaccharides In Glucan Preparations                                      Preparation Whole Glucan                                                                        Manner's                                        Contaminant Particles (strain A364A)                                                                       Glucan                                           ______________________________________                                        Glycogen    1                9.4                                              Chitin      1                2.9                                              ______________________________________                                    

Manner's preparation contains approximately 10-fold higher glycogen and3-fold higher chitin levels.

The higher protein content in Manners' preparation is a direct result ofthe extraction procedure used. Manners' process does not result inadequate hydrolysis and removal of the protein. The increased glycogenand chitin is due to two factors. The primary factor is the age of theyeast cells found in commercial baker's yeast, which is in thestationary growth phase. In the present process the growth rate of theyeast cell is closely monitored, and harvested in the late-logarithmicphase, and before the cells enter the stationary phase. The secondfactor the extraction procedure. Table 6 illustrates the significantcompositional differences resulting from Manners' procedure and theApplicants' procedure:

                  TABLE 6                                                         ______________________________________                                        Composition of Glucans                                                                  Whole Glucan                                                                  Particles                                                                     (A364A)         Manner's Glucan                                     Component   mg     %          mg    %                                         ______________________________________                                        Glucan      1000   96.2       1000  51.8                                      Protein       8    0.8        351   18.2                                      Glycogen     31    2.9        579   30.0                                      ______________________________________                                    

On a basis of 1000 mg pure glucan, the whole glucan material produced bythe process of the invention contains only 39 mg of contaminants ascompared to 930 mg for the Manners' preparation. This contaminant massdisplaces its weight in water from the glucan particles resulting in aconsiderably lower water holding capacity.

The present process results in a glucan product substantially differentthan that obtained from the procedure described by Manners, et al. TheManners product has much higher levels of protein and glycogencontamination, which results in a glucan product which lacks the waterholding properties of the Applicants' product. In addition, the resultsclearly indicate that Manners' procedure produces in an impure glucanproduct, which is only about fifty-two (52%) percent glucan. The presentprocedure yields a glucan product of about ninety-six (96%) percentpurity (see Table 6 above).

EXAMPLE 2

This example concerns the acid treatment of Saccharomyces cerevisiaeA364A whole glucan using acetic acid. A 500 mg sample of whole glucanfrom S. cerevisiae A364A produced by the method of Example 1 wassuspended in 250 milliliters of 0.5M acetic acid. The suspension wascontinuously stirred at 90° C. for 3 hours. At the end of thisextraction, the remaining insoluble glucan residue was recovered bybatch centrifugation at 5000 rpm for 20 minutes. The glucan residue waswashed once in 200 milliliters distilled water, once in 200 millilitersdehydrated ethanol and twice in 200 milliliters dehydrated ethyl ether.The resulting slurry was dried in air at 37° C. for 12 hours. Theinitial suspension in acetic acid and the supernatant were assayed fortotal carbohydrate to determine the proportion of the extractable β(1-6)glucan component. The white glucan powder obtained after drying wasresuspended in distilled water to determine its viscosity profile.Chemical modification of A364A glucan by acetic acid had aninsignificant effect on the viscosity characteristics. However,measurement of the elastic modulus of the glucan matrices showed thattheir structural rigidity can be controlled by the extent of the aceticacid extraction. FIG. 2 illustrates the effect of the acetic extractionon the structural rigidity of whole glucan derived from strain A364A andcompares this to glucan extracted from strain R4.

EXAMPLE 3

A 500 milligram sample of whole glucan from Saccharomyces cerevisiae 374produced by the method of Example 1 was suspended in 250 milliliters,0.5M acetic acid. An identical procedure to that outlined in Example 2was followed. In this case, extraction in hot acetic acid caused anincrease in the thickening properties of the glucan as shown in Table 7.

                  TABLE 7                                                         ______________________________________                                        The Viscosity of a Suspension of 374 Whole Glucan                             Compared to 374 Glucan After Acetic Acid                                      Extraction for a Range of Concentrations                                                    Viscosity in Centipoise, 25° C.                          Source of Glucan                                                                              2%     2.5%     3%   3.5%                                     ______________________________________                                        374 (Whole Glucan)                                                                            1.5    2.8       7.1  14.2                                    374 (After Extraction)                                                                        1.6    4.7      58.3 1879.0                                   ______________________________________                                    

EXAMPLE 4

A 500 milligram sample of 377 whole glucan produced by the method ofExample 1, was suspended in 150 milliliters, 0.5M acetic acid. Anidentical procedure to that outlined in Example 2 was followed.

The effect of this process on the viscosity profile of 377 glucan isshown in Table 8.

                  TABLE 8                                                         ______________________________________                                        The Viscosity of a Suspension of 377 Whole Glucan                             Compared to 377 Glucan After Acetic Acid                                      Extraction for a Range of Concentration                                                     Viscosity in Centipoise, 25° C.                          Source of Glucan                                                                              2%     2.5%     3%   3.5%                                     ______________________________________                                        377 (Whole Glucan)                                                                            1.6    3.4      16.0  75.0                                    377 (After Extraction)                                                                        1.7    4.7      58.3 1878.0                                   ______________________________________                                    

EXAMPLE 5

A 500 milligram sample of whole glucan from Saccharomyces cerevisiae R4produced by the method of Example 1 was suspended in 250 milliliters,0.5M acetic acid. An identical procedure to that outlined in Example 2was followed.

In this case the whole glucan sample before modification, has anidentical viscosity profile to the whole glucan from strain A364A;however, after the extraction the thickening properties are enhancedconsiderably, as shown in Table 9.

                  TABLE 9                                                         ______________________________________                                        The Viscosity of a Suspension of R4 Whole Glucan Compared                     to R4 Glucan After Acid Extraction for a                                      Range of Concentrations                                                                  Viscosity in Centipoise, 25° C.                             Source of Glucan                                                                           2%     2.5%    3%    3.5%   3.7%                                 ______________________________________                                        R4 (Whole Glucan)                                                                          1.8    2.3     3.1    4.5    5.4                                 R4 (After Extraction)                                                                      2.6    4.6     12.4  106.3  636.7                                ______________________________________                                    

The effect of the acetic acid treatment on the hydrodynamic propertiesof the glucan extracted from strains A364A, 374, 377 and R4, asdetermined by the linear model, is summarized in Table 10.

                  TABLE 10                                                        ______________________________________                                        Hydrodynamic Properties of Glucan After Acetic Acid Extraction                        Regression                                                                              Shape     Hydrodynamic                                              Coefficient                                                                             Factor    Volume                                            Sample  r         v         v (dl/g)  φ.sub.m                             ______________________________________                                        A364A   0.9999    2.5       0.106     0.46                                    Whole                                                                         Glucan                                                                        A364A   0.9974    2.5       0.103     0.47                                    After                                                                         Extn.                                                                         374     0.9981    4.1       0.088     0.60                                    Whole                                                                         Glucan                                                                        374     0.9995    4.1       0.103     0.44                                    After                                                                         Extn.                                                                         377     0.9997    4.1       0.091     0.45                                    Whole                                                                         Glucan                                                                        377     0.9995    4.1       0.103     0.44                                    After                                                                         Extn.                                                                         R4      0.9995    2.5       0.087     0.66                                    Whole                                                                         Glucan                                                                        R4      0.9988    2.5       0.103     0.44                                    After                                                                         Extn.                                                                         ______________________________________                                         φ.sub.m = Maximum packing fraction                                   

EXAMPLE 6 Treatment of whole glucan from Saccharomyces cerevisiae A364Awith Laminarinase

A 400 milliliter solution containing 1 milligram/milliliter whole glucanproduced by the method of Example 1, and 0.25 milligram/milliliterLaminarinase (endo β(1-3) glucanase) was prepared in phosphate buffer atpH 7.0. The solution was incubated at 37° C. for 4 hours. At the end ofthe incubation the solution was held at 70° C. for 15 minutes todeactivate the enzyme. The remaining residue was recovered bycentrifugation at 5000 rpm for 20 minutes. The resulting glucan residuewas diluted into range of concentrations in order to obtain viscositymeasurements of the Laminarinase-degraded glucan sample. Since theenzyme cannot be effectively removed from solution, a control experimentwas performed as above where the incubated enzyme contained no glucan.These readings were then used to correct the solvent viscosityaccounting for the contribution of the enzyme to the macroscopicviscosity of the suspension. The effect of this process on the viscosityprofile of an A364A glucan suspension is shown in FIG. 3, and in Table11.

Table 11 shows the pronounced effect of the enzyme treatment on theglucan. At a concentration of 3.7% w/v an 80-fold decrease in theviscosity has been achieved.

                  TABLE 11                                                        ______________________________________                                        The Viscosity of Suspension of A364A Glucan                                   Before and After the Enzyme Modification for a Range                          of Concentrations                                                                         Viscosity in Centipoise, 25° C.                            Source of Glucan                                                                            1%      2%      3%   3.5%  3.7%                                 ______________________________________                                        A364A (Whole Glucan)                                                                        1.4     2.7     13.7 127.3 816.3                                A364A (After Enzy-                                                                          1.2     1.6      3.1  6.4   10.2                                matic Digestion                                                               ______________________________________                                    

EXAMPLE 7 Treatment of Whole Glucan From Saccharomyces cerevisiae 374with Laminarinase

A sample of 374 whole glucan was subjected to the process outlined indetail in Example 6. This process caused a decrease in the thickeningproperties of this glucan preparation as shown in Table 12.

                  TABLE 12                                                        ______________________________________                                        The Viscosity of Suspension of 374 Glucan                                     Before and After the Enzyme Modification for a Range                          of Concentrations                                                                         Viscocity in Centipoise, 25° C.                            Source of Glucan                                                                             1%     2%       3%   3.5%                                      ______________________________________                                        374 (Whole Glucan)                                                                           1.6    4.0      47.4 2630.7                                    374 (After Enzy-                                                                             1.3    2.3       9.3  62.9                                     matic Digestion)                                                              ______________________________________                                    

EXAMPLE 8 Treatment of whole glucan from Saccharomyces cerevisiae 377with Laminarinase

A sample of 377 whole glucan was subjected to the process outlined indetail in Example 5. The effect of this process on this glucanpreparation was similar to that on 374 glucan up to a concentration of2% w/v. However, at higher concentrations the viscosity of the treatedglucan was higher. This product therefore possesses extremely valuableproperties since it has a negligible effect on suspension viscosities oflow concentrations yet exhibits very high thickening properties atconcentrations above 3% w/v. Table 13 below quantitatively describes thethickening properties of this product.

                  TABLE 13                                                        ______________________________________                                        The Viscosity of Suspension of 377 Glucan                                     Before and After the Enzyme Modification for a Range                          of Concentrations                                                                          Viscocity in Centipoise 25° C.                                         1%   2%        3%     3.5%                                       ______________________________________                                        377 (Whole Glucan)                                                                           1.6    3.4       16.0  75.0                                    377 (After Enzy-                                                                             1.4    2.8       18.1 367.4                                    matic Digestion                                                               ______________________________________                                    

The effect of this enzyme digest on the hydrodynamic properties of theglucan samples from strains A364A, 374, and 377 is summarized in Table14.

                  TABLE 14                                                        ______________________________________                                        Hydrodynamic Properties of Glucan                                             After Laminarinase Digest                                                             Regression                                                                              Shape     Hydrodynamic                                              Coefficient                                                                             Factor    Volume                                            Sample  r         V         v (dl/g)  φ.sub.m                             ______________________________________                                        A364A   0.9999    2.5       0.106     0.46                                    Whole                                                                         Glucan                                                                        A364A   0.9998    2.5       0.057     0.27                                    After                                                                         Digest                                                                        374     0.9987    4.1       0.088     0.36                                    Whole                                                                         Glucan                                                                        374     0.9985    4.1       0.070     0.30                                    After                                                                         Digest                                                                        377     0.9974    4.1       0.091     0.45                                    Whole                                                                         Glucan                                                                        377     0.9989    4.1       0.065     0.27                                    After                                                                         Digest                                                                        ______________________________________                                         φ.sub.m = Maximum packing fraction                                   

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

We claim:
 1. Whole glucan particles isolated from glucan-containing cellwalls, the isolated glucan particles substantially retaining the in vivoglucan morphology, said whole glucan particles having β(1-3) linkagessufficiently altered to provide a significant difference in thehydrodynamic volume of said altered whole glucan particles compared tothe hydrodynamic volume of non-altered whole glucan particles. 2.Altered whole glucan particles of claim 1 wherein the glucan particlesare isolated from yeast cells.
 3. Altered whole glucan particles ofclaim 2 wherein the glucan particles are isolated from a strain of yeastselected from the group consisting of: Saccharomyces cerevisiae,Saccharomyces delbrueckii, Saccharomyces rosei, Saccaromycesmicroellipsodes, Saccharomyces carlsbergensis, Saccharomyces bisporus,Saccharomyces fermentati, Saccharomyces rouxii, Schizosaccharomycespombe, Kluyveromyces lactis, Kluyveromyces fragilis, Kluyveromycespolysporus, Candida albicans, Candida cloacae, Candidatropicalis,Candida utilis, Hansenula wingei, Hansenula arni, Hansenula henricii,Hansenula americana, Hansenula canadiensis, Hansenula capsulata,Hansenula polymorpha, Pichia Kluyveri, Pichia pastoris, Pichiapolymorpha, Pichia rhodanensis, Pichia ohmeri, Torulopsis bovina andTorulopsis glabrata.
 4. Altered whole glucan particles of claim 3wherein the whole glucan particles are isolated from a strain ofSaccharomyces cerevisiae yeast.
 5. Altered whole glucan particles ofclaim 4 wherein the strain of Saccharomyces cerevisiae yeast isSaccharomyces cerevisiae A364A, Saccharomyces cerevisiae 374,Saccharomyces cerevisiae 377, Saccharomyces cerevisiae R4 NRRL Y-15903or a mixture thereof.
 6. Altered whole glucan particles of claim 5containing less than one percent, by weight, protein.
 7. Altered wholeglucan particles of claim 1 isolated from a strain of yeast geneticallymodified to produce altered β(1-3) linkages.
 8. A process for preparingwhole glucan particles having altered β(1-3) linkages and havingsubstantially the in vivo morphology of glucans, comprising:a)extracting non-glucan components from glucan-containing cell wallswithout a prior step of disrupting the cell walls to thereby producewhole glucan particles substantially retaining the in vivo glucanmorphology; and b) treating said whole glucan particles obtained in step(a) under conditions appropriate to substantially alter β(1-3) linkagesin said whole glucan particles to provide a significant difference inthe hydrodynamic volume of the altered whole glucan particles comparedto the hydrodynamic volume of the non-altered isolated whole glucanparticles.
 9. A process of claim 8 wherein said treatment of step (b)comprises contacting the whole glucan particles with a glucanase at a pHof from about 5 to about 8 and at a temperature of from about 20° C. toabout 60° C.
 10. A process of claim 9 wherein the glucanase compriseslaminarinase.
 11. A process of claim 8 wherein the treatment of step (b)comprises contacting the whole glucan particles with an acid.
 12. Aprocess of claim 11 wherein the extraction of step (a) is performed bycontacting the glucan-containing cells with an aqueous hydroxidesolution having a normality of from about 0.1 to about
 10. 13. A processof claim 12 wherein the aqueous hydroxide solution comprises an aqueoussolution of sodium hydroxide.
 14. A process of claim 13 wherein theglucan-containing cell walls comprise yeast cell walls and the yeast isselected from a strain of yeast from the group consisting of:Saccharomyces cerevisiae, Saccharomyces delbrueckii, Saccharomycesrosei, Saccaromyces microellipsodes, Saccharomyces carlsbergensis,Saccharomyces bisporus, Saccharomyces fermentati, Saccharomyces rouxii,Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis,Kluyveromyces polysporus, Candida albicans, Candida cloacae,Candidatropicalis, Candida utilis, Hansenula wingei, Hansenula arni,Hansenula henricii, Hansenula americana, Hansenula canadiensis,Hansenula capsulata, Hansenula polymorpha, Pichia Kluyveri, Pichiapastoris, Pichia polymorpha, Pichia rhodanensis, Pichia ohmeri,Torulopsis bovina and Torulopsis glabrata.
 15. A process of claim 13wherein the yeast cells are a strain of Saccharomyces cerevisiae.
 16. Aprocess of claim 15 wherein the strain of Saccharomyces cerevisiae isSaccharomyces cerevisiae A364A, Saccharomyces cerevisiae 374,Saccharomyces cerevisiae 377 or a mixture thereof.
 17. Altered wholeglucan particles produced by a process of claim
 8. 18. Altered wholeglucan particles produced by a process of claim
 9. 19. Altered wholeglucan particles produced by a process of claim 11.